Graduate Program

Biological Sciences

Degree Name

Master of Science (MS)

Semester of Degree Completion

2013

Thesis Director

Steven L. Daniel

Thesis Committee Member

Gary A. Bulla

Thesis Committee Member

Kai Hung

Abstract

Compared to the other parts of the human body, the gastrointestinal tract harbors a complex microbiota. The impact of the indigenous microbiota on host physiology is most pronounced in the colon, where the primary bile acids chenodeoxycholic acid and cholic acid are converted, via 7α-dehydroxylation, to the toxic secondary bile acids lithocholic acid and deoxycholic acid, respectively, by Clostridium scindens, an obligate anaerobe. Interestingly, other than bile acid dehydroxylation, little is known about the basic physiology of C. scindens. Understanding the metabolism of C. scindens will hopefully provide much-needed information as to how this gut bacterium impacts human health and disease. The goals of our study were to determine the vitamin and amino acid requirements of C. scindens and also to determine if C. scindens can convert primary bile acid cholic acid to secondary bile acid deoxycholic acid via 7α-dehydroxylation under defined culture conditions. C. scindens VPI 12708 was routinely maintained in anaerobic BHI broth medium at 37°C. When C. scindens was transferred from the BHI medium to a defined medium (DM; 25 mM glucose, minerals, metals, bicarbonate, 100% CO2 gas phase, and cysteine), growth was negligible. Only when DM was supplemented with a vitamin mix (p-aminobenzoate, biotin, cyanocobalamin, folate, lipoate, nicotinate, pantothenate, pyridoxal, riboflavin, and thiamine) and an amino acid mix (alanine, arginine, asparagine, aspartate, cystine, glutamate, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine) was growth observed. The leave-one-out technique was subsequently used to determine the specific vitamin(s) and amino acid(s) required for growth. With this technique, two vitamins (riboflavin and pantothenate) and one amino acid (tryptophan) were found to be essential for the growth of C. scindens. Growth was also reduced, but not inhibited when pyridoxal or biotin was absent from OM. It was also observed that under defined culture conditions C. scindens was able to convert cholic acid to deoxycholic acid via 7α-dehydroxylation while producing two unknown products.

Download

Included in

Bacteriology Commons

Share

COinS